A

pwsasbfcSLpe©

K

f

0

Journal of Biotechnology 126 (2006) 123–134

Molecular identification of the edible ectomycorrhizalfungus Lactarius deliciosus in the symbiotic

and extraradical mycelium stages

Sara Hortal, Joan Pera, Luis Galipienso, Javier Parlade ∗

IRTA, Departament de Proteccio Vegetal, Ctra. Cabrils s/n, 08348 Cabrils, Barcelona, Spain

Received 22 December 2005; received in revised form 22 March 2006; accepted 7 April 2006

bstract

Specific rDNA ITS amplifications, microsatellite-primed PCR and ITS-SSCP analysis were applied to identify and characterizere-selected isolates of the edible ectomycorrhizal fungus Lactarius deliciosus in different stages of the life cycle. Samplingas performed from pure cultures, mycorrhizas and soil from experimental plots established with nursery-inoculated pine

eedlings. A newly-designed reverse primer (LDITS2R) combined with the universal forward ITS1 allowed to perform specificmplifications of L. deliciosus from all the samples. Microsatellite-primed PCR using the (GTG)5 oligonucleotide as a primerhowed clear polymorphisms among the different L. deliciosus isolates. The patterns of mycorrhiza samples showed additionalands corresponding to the plant DNA. Single strand conformation polymorphism (SSCP) analysis of the specific rDNA ITSragment amplified from 18 L. deliciosus isolates showed nine clearly different patterns. Mycorrhiza and soil samples showedoincident patterns with their respective fungal isolates. Specific rDNA ITS amplifications had not been previously used forSCP analysis of ectomycorrhizas and extraradical mycelium. This relatively simple and inexpensive technique allows tracking

. deliciosus isolates in different stages of the fungus development. Specific ITS-SSCP analysis is promising in studies of theersistence of inoculated L. deliciosus isolates and their competitiveness with native ectomycorrhizal fungi, especially at thextraradical mycelium stage.

2006 Elsevier B.V. All rights reserved.

TS-SSC

eywords: Edible ectomycorrhizal fungi; Genomic fingerprinting; I

∗ Corresponding author. Tel.: +34 93 750 75 11;ax: +34 93 753 39 54.

E-mail address: xavier.parlade@irta.es (J. Parlade).

1

o1rc

168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2006.04.011

P; Lactarius; Microsatellite-primed PCR; Specific primers

. Introduction

Lactarius is one of the larger known genus

f ectomycorrhiza-forming basidiomycetes (Singer,986; Hutchison, 1999). The several hundred taxa cur-ently recognized play an important role as late-stageolonizers of a variety of woody trees and shrubs in a

1 iotechn

w1eehiClsinwmieacoccHmsPmwbwTtTiiss1

fifi(to1aamr

i2Heuorsu1tuiaP(gvbcai(ea(lrfA

(tctticcbmtaS

24 S. Hortal et al. / Journal of B

ide range of ecosystems (Trappe, 1962; Hutchison,999). Some species produce commercially valuabledible fruit bodies. Key species as L. deliciosus (L.x Fr.) S.F. Gray and L. sanguifluus (Paulet ex) Fr. areighly esteemed and there is a valuable trade, especiallyn local markets of Europe (Singer, 1986; FAO, 2004).ommercial harvesting of edible fungi is an important

ocal business in many countries. The export trade ofome species, as in the case of the edible Lactarius spp.,s driven by a strong and expanding demand, which can-ot be supported by forest harvesting in a sustainableay (Hall and Wang, 1998; FAO, 2004). Managingycorrhizal inoculations with edible Lactarius spp. to

ncrease and diversify forest productivity is a promisingconomic alternative for many Mediterranean forestreas with limited resources. Nevertheless, the diffi-ulties in managing pure cultures of most edible myc-rrhizal fungi and producing mycorrhizal plants underontrolled conditions have limited the development ofontrolled mycorrhizal inoculations (Hutchison, 1999;all et al., 2003). Guerin-Laguette et al. (2000) opti-ised the experimental conditions to produce Pinus

ylvestris L. plants mycorrhizal with L. deliciosus.arlade et al. (2004) evaluated different inoculationethods of Pinus pinaster and P. sylvestris seedlingsith L. deliciosus under greenhouse conditions. Theest percentages of root colonization were obtainedith vegetative inoculum in a peat–vermiculite matrix.he same method was used by Rincon et al. (1999)

o obtain mycorrhizas of L. deliciosus + P. pinea L.he establishment of field plantations with Lactarius-

noculated P. pinaster Ait. seedlings to obtain fruitbod-es was first attempted by Poitou et al. (1984). Furthertudies on these plantations showed the production ofporocarps after 3 years in the field (Guinberteau et al.,989).

Outplanting of inoculated tree seedlings into theeld raises the problem to identify the introducedungus at species and isolate level and to determinets persistence and competitivity with native fungiSelosse et al., 1998). Assessment of sporocarp produc-ion and mycorrhizal morphotypes can be performedver time (Villeneuve et al., 1991; Le Tacon et al.,992). However, many of the morphological features

re not conclusive for the identification of mycorrhizast species level. In this sense, the development ofolecular techniques based on the polymerase chain

eaction (PCR) represented a significant step in the

niHh

ology 126 (2006) 123–134

dentification of ectomycorrhizas (Di Battista et al.,002; Gardes et al., 1991; Gardes and Bruns, 1993;enrion et al., 1992; Horton and Bruns, 2001; Selosse

t al., 1998; Weber et al., 2002). Different molec-lar approaches are currently available in the studyf genetic polymorphisms with a variable degree ofesolution. Amplification of the internal transcribedpacer (ITS) regions in the ribosomal genes (rDNA)sually reveal interspecific variations (Bruns et al.,991; Gomes et al., 2002; Horton, 2002). Also, iden-ification of individual genets is essential for a betternderstanding of the diversity, structure, and dynam-cs of populations of ectomycorrhizal fungi (Martin etl., 1998). Microsatellite-primed PCR, or interrepeatCR, and single strand conformation polymorphismSSCP) analysis are two powerful and sensitive fin-erprinting approaches to detect intraspecific geneticariability. Microsatellite-primed PCR was introducedy Meyer et al. (1993). In this technique, oligonu-leotides complementary to microsatellites are useds single primers to amplify interrepeat sequencesn a wide range of animal, plant and fungal speciesMartin et al., 1998). Complex fingerprints are gen-rated, allowing detection of polymorphisms at inter-nd intraspecific levels and subsequent identificationStenlid et al., 1994). From the several microsatel-ite primers screened, the sequence (GTG)5 has beeneported to provide a high polymorphism degree in dif-erent species of symbiotic fungi (Martin et al., 1998;micucci et al., 2001).Single strand conformation polymorphism analysis

SSCP) is a technique based on the differences in elec-rophoretic mobility of single-stranded DNA due toonformational changes (Orita et al., 1989). Whereashe mobility of double-stranded DNA in gel elec-rophoresis depends on strand length and is relativelyndependent of the nucleotide sequence, single-strandonformations are noticeably affected by very smallhanges in the sequence. The technique is reported toe capable of detecting 99% of point mutations in DNAolecules of 100–300 bp in length and 89% muta-

ions in molecules of 300–450 bp in length (Orita etl., 1989; Mohabeer et al., 1991; Hayashi, 1992). TheSCP technique is one of the most widely used tech-

iques for the identification of point mutations due tots simplicity, sensitivity and rapidity (Orita et al., 1989;ayashi, 1991, 1992; Nair et al., 2002). This techniqueas been applied for resolving genotypes and assessing

iotechn

tf

timftr

2

2a

oBPted

pwbi(ossiipcssavwtpvL((a

1swFp

2

atGE(gpcot

PCs

2p

ftsG3MDHa

9Cffaa

S. Hortal et al. / Journal of B

he reproductive genetics of natural ectomycorrhizalungal populations (Bonello et al., 1998).

The objective of this study is to detect consis-ent polymorphisms useful to effectively character-ze and identify L. deliciosus species and isolates by

icrosatellite-primed PCR and ITS-SSCP analysis ofungal DNA samples obtained from different stages ofhe symbiosis: pure cultures from sporocarps, mycor-hizas and rhizospheric soil from experimental plots.

. Materials and methods

.1. Fungal sampling: cultures, ectomycorrhizasnd rhizospheric soil

Fungal cultures of different Lactarius species werebtained by plating excised tissue of fresh sporocarps inAF medium (Oort, 1981) as described in Molina andalmer (1982). Cultures were maintained at 4 ◦C and

ransferred onto fresh medium every 3 months. Ref-rences of cultures, voucher specimens and collectionata are shown in Table 1.

Ectomycorrhizas formed by L. deliciosus + P.inaster and by L. deliciosus + P. canariensis Smithere obtained axenically in synthesis tubes as reportedy Molina (1979) with slight modifications consistingn the replacement of MMN by BAF liquid mediumParlade et al., 2004). Fungal inoculations were carriedut just after seed germination with 5 ml of myceliallurries prepared by blending 1-month old colonies interile distilled water (approximately 0.03 g mycelium,n dry weight, per plant) of each of the followingsolates: 312, 120, 330, 331, 332, 178, 313 (for P.inaster inoculations) A34C, A49C, and A61C (for P.anariensis inoculations) (Table 1) which were pre-elected for their colonization ability in pure cultureynthesis (data not shown). The mycorrhizal associ-tion Pinus canariensis + L. deliciosus was not pre-iously reported. Rhizospheric soil and mycorrhizasere collected in October 2004 from a field planta-

ion established in December 2002 with 1-year old P.inea L. seedlings inoculated in the greenhouse withegetative inoculum of the isolates 312 and 178 of

. deliciosus (Table 1) as described in Parlade et al.2004). Average mycorrhizal colonization of the plantspercentage of short roots colonized with L. deliciosus)t outplanting was 60 and 37% for the isolates 312 and

ebpe

ology 126 (2006) 123–134 125

78, respectively. Rhizospheric soil was obtained byhaking the root system of unburied plants. The soilas then sieved with a 2 mm mesh to remove roots.ield mycorrhizas were also obtained from the samelants.

.2. DNA extractions

Genomic DNA from fungal colonies, mycorrhizasnd uncolonized short roots was obtained by usinghe DNeasy® Plant Mini Kit (Qiagen GmbH, Hilden,ermany) following the manufacturer’s instructions.xtractions from agar cultures were made from 10 mg

fresh weight) of fungal material taken from the mar-in of the colony. Branched mycorrhizas were sam-led and processed as the mycelium from fungalolonies, except soil mycorrhizas which were previ-usly surface-sterilized with 30% H2O2 for 30 s andhen rinsed with sterile distilled water.

Rhizospheric soil DNA extraction was done with theowerSoilTM DNA Isolation Kit (MoBio Laboratories,arlsbad, CA, USA) from 0.25 g soil (fresh weight) per

ample according to the manufacturer’s instructions.

.3. ITS amplifications, sequencing and specificrimers design

Amplifications of the rDNA ITS region from all theungal isolates were obtained by PCR reactions con-aining 2 �l (from a 10 �M stock) of each of the univer-al primers ITS1 (5′-TCC GTA GGT GAA CCT GCG-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-′) (White et al., 1990), 20 �l of Eppendorf® Master-ix 2.5X (Eppendorf AG, Hamburg, Germany), 2 �l ofNA template (corresponding to 20–40 ng DNA) andPLC water (Scharlau-Chemie, Barcelona, Spain) tofinal volume of 50 �l.

PCR reactions were performed in a GeneAmp®

700 thermocycler (Applied Biosystems, Foster City,A, USA) with an initial denaturation step of 95 ◦C

or 3 min, followed by 35 cycles of amplification (40or soil samples) at 95 ◦C for 20 s, 55 ◦C for 30 snd 72 ◦C for 40 s, and a final extension of 5 mint 72 ◦C. Amplification products were separated by

lectrophoresis in 2% agarose gels in 0.5× TBEuffer (1× TBE = 0.09 M Tris-borate, 0.002 M EDTAH 8) and visualized on a UV-transilluminator afterthidium bromide staining. The amplification prod-

126 S. Hortal et al. / Journal of Biotechnology 126 (2006) 123–134

Table 1Lactarius isolates used in this study

Fungal species Isolate numbera Herbariumnumbera

Associated treespecies

Collection date Location GenBankAccession no.b

L. chrysorrheus 262 569 P. sylvestris 19/10/1995 Girona –L. deliciosus 120 246 P. pinaster 16/11/1988 Girona DQ116894L. deliciosus 178 364 P. pinea 27/10/1994 Barcelona DQ116888L. deliciosus 217 444 P. pinea 19/11/1994 Girona DQ116895L. deliciosus 274 612 P. pinea 24/10/1995 Girona DQ116889*a

DQ116890*bL. deliciosus 312 669 P. pinaster 24/10/2000 Girona DQ116886L. deliciosus 313 670 P. pinea 24/10/2000 Girona DQ116887L. deliciosus 330 687 P. halepensis 30/11/2003 Barcelona DQ116898L. deliciosus 331 688 P. sylvestris 12/10/2003 Tarragona DQ116899L. deliciosus 332 689 P. sylvestris 12/10/2003 Barcelona DQ116900L. deliciosus 334 691 P. sylvestris 26/09/2004 Barcelona DQ116901L. deliciosus 335 692 P. sylvestris 26/09/2004 Barcelona DQ116902L. deliciosus 336 693 P. sylvestris 12/10/2004 Barcelona DQ116903L. deliciosus 337 694 P. sylvestris 12/10/2004 Barcelona DQ116904L. deliciosus A34C A34H P. canariensis 21/01/2003 G. Canaria DQ116893L. deliciosus A49C A49H P. canariensis 03/12/2003 G. Canaria DQ116896L. deliciosus A61C A61H P. canariensis 04/12/2003 G. Canaria DQ116897L. deliciosus 892 892 P. sylvestris 26/09/2002 Soria DQ116892L. deliciosus 894 894 P. sylvestris 27/09/2002 Soria DQ116891L. rufus 344 700 P. uncinata 15/10/2004 Andorra DQ116912L. sanguifluus 261 550 P. radiata 19/10/1995 Girona DQ116905L. sanguifluus 263 580 P. pinaster 24/10/1995 Girona DQ116906L. sanguifluus 328 685 P. halepensis 06/11/2003 Barcelona DQ116907L. sanguifluus 329 686 Pinus sp. 19/10/2003 Teruel DQ116909L. sanguifluus 333 690 P. pinea 01/11/2003 Barcelona DQ116908L. semisanguifluus 338 695 P. uncinata 15/10/2004 Andorra DQ116910L. semisanguifluus 339 696 P. uncinata 15/10/2004 Andorra DQ116911L. tesquorum 345 701 Cistus sp. 21/12/2004 Girona DQ116913

A laborces from

uPSbBsfiCaaw(cT2U

Imci

facto

2

a Cultures and voucher herbarium specimens are deposited at IRTb GenBank accession numbers for the rDNA ITS region. *Sequen

cts of each isolate were purified with a Roche®Highure PCR Product Purification Kit (Roche Appliedcience, Indianapolis, IN, USA), and sequenced inoth directions using a 3730 DNA Analyzer (Appliediosystems, Foster City, CA, USA). When consensus

equences showed many indeterminations, the ampli-ed ITS products were cloned in pGEM-T-Easy DNAloning Vector System (Promega, Madison, WI, USA)ccording to the protocol described by Sambrook etl. (1989) and sequenced. The alignment of sequencesas performed with the BioEdit program version 5.0.9

Hall, 1999). A reverse specific primer for L. deli-

iosus named LDITS2R (5′-AGA GGA GCT GGGCT AAG-3′) was designed with the Primer Express.0 software (Applied Biosystems, Foster City, CA,SA). Specific amplifications with the pair of primers

oa

atory.clones ‘a’ and ‘b’.

TS1/LDITS2R were attempted from pure cultures,ycorrhizas and rhizospheric soil samples. The PCR

onditions with this pair of primers were modified byncreasing the annealing temperature to 62 ◦C.

Microsatellite-primed PCR reactions were per-ormed using the same amplification mix as describedbove and 4 �l (from a 10 �M stock) of the oligonu-leotide (GTG)5 as a unique primer. Genomic DNAemplates from pure cultures, mycorrhizas and uncol-nized roots of the host plants were included.

.4. SSCP analysis

Specific ITS1/LDITS2R amplification productsbtained from L. deliciosus isolates, mycorrhizasnd rhizospheric soil samples were analysed for sin-

iotechn

gmrtos1c4bb

3

3

wIfsn(wtpStwris

a9Htbngaww(swd

taficcSefcIlpanIadcms

3ia

a4rbctonss

wdbsus

S. Hortal et al. / Journal of B

le strand conformation polymorphisms (SSCP). Twoicrolitres of amplified DNA from each isolate (cor-

esponding to approximately 4 �g DNA) were dena-ured at 95 ◦C for 5 min in formamide buffer, chilledn ice for 5 min, and then resolved by electrophore-is in non-denaturing 10% polyacrylamide gels in× TBE buffer. Electrophoresis was carried out atonstant voltage of 250 V for 2 h and 30 min at◦C. The gels were stained with silver nitrate andands visualized according to the procedure describedy Beidler et al. (1982).

. Results

.1. Specific identification of L. deliciosus by PCR

PCR products of approximately 650 bp amplifiedith the ITS1/ITS4 primers and corresponding to the

TS1, 5.8S and ITS2 regions of the rDNA were obtainedrom all the dikariontic isolates (Table 1). Consensusequences of heterozygotic isolates showed punctualucleotide changes or simple insertion and deletionindel) mutations in both ITS1 and ITS2 regions, whichere clearly identified in the chromatograms. However,

he isolate 274 was cloned due to the presence of super-osed signals and the high number of indeterminations.ingle strand conformation polymorphism analysis of

he clones showed two different banding patterns whichere named ‘a’ and ‘b’ (Table 1). Posterior sequencing

evealed two indel mutations involving ‘T’ nucleotidesn the positions 16 and 187 (in the submitted GenBankequences).

Sequence alignments showed identities over 99%mong isolates belonging to L. deliciosus and around2% between L. sanguifluus or L. semisanguifluuseim e Lecl. and L. deliciosus isolates. Sequence iden-

ities of L. deliciosus with other Lactarius species wereelow 90%. An area with a consistent insertion of 8–11ucleotides in the ITS2 region was detected in L. san-uifluus isolates, compared to L. deliciosus ones. Thisrea was variable enough among Lactarius species andas used to design the primer LDITS2R (reverse),hich was combined with the universal primer ITS1

forward) for L. deliciosus specific amplifications. Aearch for short exact matches with the specific primeras performed in the GenBank database and only L.eliciosus entries showed a 100% identity.

tifT

ology 126 (2006) 123–134 127

The specificity of the primers ITS1/LDITS2R wasested with all the Lactarius isolates shown in Table 1,s well as with DNA extractions from cultures ofve common species of ectomycorrhizal fungi (Lac-aria laccata (Scop.:Fr.) Berk. & Broome, Hebelomarustuliniforme (Bull.) Quel., Boletus edulis Bull.:Fr.,phaerosporella brunnea (Alb. et Schwein.:Fr.) Svrcekt Kubicka and Thelephora terrestris Ehrenb.:Fr.) androm non-mycorrhizal pine roots. Parallel amplifi-ations were performed with the universal primersTS1/ITS4 as positive controls. Only L. deliciosus iso-ates were successfully amplified by PCR with therimers ITS1/LDITS2R whereas all the isolates weremplified with the universal primers ITS1/ITS4 (dataot shown). Specific amplifications with the primersTS1/LDITS2R were also obtained from mycorrhizasnd rhizospheric soil samples from outplanted L.eliciosus-inoculated seedlings. Amplification bandsorresponding to the different samples (pure cultures,ycorrhizas and rhizospheric soil) showed a similar

ize of around 500 bp (Fig. 1).

.2. Intraspecific identification of L. deliciosussolates by microsatellite-primed PCR and SSCPnalysis

The (GTG)5 microsatellite primers yielded a vari-ble number of amplification products ranging from00 to 1200 bp (Fig. 2). The amplification fingerprintsevealed highly polymorphic interrepeat sequencesetween isolates and similar patterns between pureultures 312, 178, A61C and 330 and their respec-ive mycorrhizas. Also, a faint banding pattern wasbserved in amplifications of plant DNA from uncolo-ized short roots. Amplification fingerprints were con-istently reproduced in repeated experiments (data nothown).

SSCP analyses of the specific ITS amplificationsith the primers ITS1/LDITS2R for each of the L.eliciosus isolates provided a pattern of two to fourands with clear polymorphisms (Fig. 3). Groupingimilar banding patterns allowed the differentiation ofp to nine different patterns among the 18 L. delicio-us isolates. Identical patterns grouped isolates with

he same sequences as well as isolates, which differedn one or more nucleotides. On the other hand, dif-erent patterns showed at least one nucleotide change.he alignment of the amplified sequences from all

128 S. Hortal et al. / Journal of Biotechnology 126 (2006) 123–134

Fig. 1. Specific PCR products amplified with the primers ITS1-LDITS2R in different stages of the fungal symbiosis: mycorrhizas samples(represented in the left gel by the isolate number + host plant) and rhizosphere samples in the right gel (Rhiz followed by the number of theinoculated isolate). Positive controls were pure cultures (isolate number alone) and soil + fungal isolate added as a pure culture. Pine, Pinuspinea; Pina, P. pinaster; Pcan, P. canariensis; Wpcr, control PCR; M, 100 bp marker.

Fig. 2. Polymorphisms of the PCR products obtained by amplification of genomic DNA with the primer (GTG)5 from different samples:Lactarius spp. isolates (pure cultures), mycorrhizas (isolate number + host plant) and pine roots. The numbers correspond to the isolates shownin Table 1. M, 100 bp marker; Wpcr, control PCR; Pine, Pinus pinea; Pina, P. pinaster.

S. Hortal et al. / Journal of Biotechn

Fig. 3. Single strand conformation polymorphisms (SSCP) for PCRproducts obtained by specific rDNA ITS amplifications (primersITS1, LDITS2R) from different Lactarius deliciosus isolates. Thenumbers correspond to the isolates shown in Table 1. Similar pat-terns share the same roman number.

tepdtc

stp(br3p(

4

ws

Table 2Single nucleotide polymorphisms (SNPs) of Lactarius deliciosus isolates ge

Isolates compareda Fragment positionb Type of SN

120/894 46 Base chang120/49 46 Base chang120/61 46 Base chang894/49 46 Base chang894/61 46 Base chang894/331 46 Base chang331/49 46 Base chang331/61 46 Base chang

336/120 92 Base chang336/331 92 Base chang120/330 92 Base chang120/313 92 Base chang313/331 92 Base chang331/330 92 Base chang

336/217 107 Base chang313/217 107 Base chang217/330 107 Base chang

120/178 217 Indel331/178 217 Indel

a Isolates numbered as in Table 1.b In the fragment of 510 bp amplified with the primers ITS1/LDITS2R; 3c Y:C or T; R:G or C.d In the SSCP pattern represented in the gel of Fig. 3.

ology 126 (2006) 123–134 129

he L. deliciosus isolates allowed identifying up toight single-nucleotide polymorphisms (SNPs). Com-arisons of sequences with only one single-nucleotideifference and having different SSCP patterns allowedo detect four positions related with the conformationhanges (Table 2).

Specific amplifications from different mycorrhizasynthesized with 10 L. deliciosus isolates and three hostree species showed identical SSCP patterns as com-ared with those of their corresponding pure culturesFig. 4) irrespective of the host tree involved in the sym-iosis. Similarly, soil DNA specific amplifications fromhizospheric soil of plants inoculated with the isolates12 and 178 showed identical pattern as the originalure cultures from which inoculations were performedFig. 5).

. Discussion

The designed reverse primer (LDITS2R) combinedith the universal ITS1 forward primer allowed the

pecific identification of L. deliciosus by PCR in dif-

nerating differences in the ITS-SSCP pattern

P Nucleotides involvedc Number of bandsd

e Y/T 3/2e Y/C 3/2e Y/C 3/2e T/C 2/2e T/C 2/2e T/Y 2/3e Y/C 3/2e Y/C 3/2

e Y/C 3/3e Y/C 3/3e C/Y 3/3e C/Y 3/3e Y/C 3/3e C/Y 3/3

e G/R 3/3e G/R 3/3e R/G 3/3

–/T 3/4–/T 3/4

0 bases less in the sequence submitted to GenBank.

130 S. Hortal et al. / Journal of Biotechn

Fig. 4. Single strand conformation polymorphisms (SSCP) for PCRproducts obtained by specific rDNA ITS amplifications (primersI(lc

fsiicDetR(M22

FpimF

rtprl(Gte2dm

uomsl(cilhc(1M

u(

TS1, LDITS2R) from Lactarius deliciosus isolates and mycorrhizasisolate number + host plant). The numbers correspond to the iso-ates shown in Table 1. Pine, Pinus pinea; Pina, P. pinaster; Pcan, P.anariensis.

erent stages of its life cycle (mycelium isolated fromporocarps, axenic and field mycorrhizas and extrarad-cal soil mycelium). Most of the recent studies on thedentification of belowground ectomycorrhizal fungalommunities and their spatio-temporal patterns rely onNA-analysis techniques (Rosling et al., 2003; Leake

t al., 2004) which generally involve total DNA extrac-ion, followed by fungal rDNA ITS amplifications andFLP analysis or sequencing, either from mycorrhizas

Guerin-Laguette et al., 2003; Rosling et al., 2003;urat et al., 2005) or soil mycelium (Chen and Cairney,

002; Landeweert et al., 2003a,b; Wallander et al.,003). Dickie et al. (2002) used PCR and terminal

ig. 5. Single strand conformation polymorphisms (SSCP) for PCRroducts obtained by specific rDNA ITS amplifications from Lactar-us deliciosus, isolates 312 and 178, in different samples: pure culture

ycelium mycorrhizas and rhizospheric soil. For abbreviations, seeig. 1.

(a2gesaiagacmDwcob

ology 126 (2006) 123–134

estriction fragment length polymorphism (T-RFLP)echniques to identify specific fungal hyphae in a soilrofile. Intraspecific variability of the intergenic spaceregion (IGS) for the identification of the fungal iso-ates forming mycorrhizas was studied by Albee et al.1996), Henrion et al. (1992), Selosse et al. (1996) anduidot et al. (1999). On the other hand, DNA quan-

ification techniques such as competitive PCR (Guidott al., 2002, 2003) and real-time PCR (Schubert et al.,003; Landeweert et al., 2003b) have also been used toetermine the distribution and persistence of the fungalycelium in the soil.The application of molecular techniques in the eval-

ation of the field persistence of a given fungal speciesr isolate has been particularly useful in controlledycorrhizal inoculations, especially for those fungal

pecies obtained after a selection process and estab-ished in long-term field experiments. Selosse et al.1998, 1999, 2001) followed the persistence of Lac-aria species and genets by RAPDs and rDNA analysesn different plantations up to 10 years after the estab-ishment of the inoculated plants. Also, specific primersave been designed for selective amplifications of espe-ially valuable fungal species as the edible B. edulisMello et al., 2006) and Tuber sp. (Amicucci et al.,998; Mello et al., 1999; Sejalon-Delmas et al., 2000;abru et al., 2001, 2004).Microsatellite-primed PCR had been previously

sed for intraspecific characterization of L. amethystinaBull.) Murrill, species of Tirmania and TerfeziaMartin et al., 1998), T. borchii Vittad. (Lanfranco etl., 1998) and different Tuber species (Amicucci et al.,001). In the present study, we have obtained distin-uishable patterns in the PCR products from the differ-nt Lactarius isolates. Also, the shared bands betweenome isolates (observed in the origins of Canary Islandsnd Soria) suggest a relationship between the band-ng patterns and the geographic origin. This fact waslso reported by Martin et al. (1998) with Terfezia fin-erprints. Some of our mycorrhiza samples showeddifferent pattern from their respective fungal pure

ultures, probably due to the presence of amplifiedicrosatellite interrepeat sequences in the host plantNA. Because of these limitations, the technique

ould be useful for analyzing pure cultures from sporo-

arps but is of uncertain utility for the characterizationf ectomycorrhizas unless a fungal pure culture coulde isolated. Actually, this technique has been used basi-

iotechn

c1WttEmiD

ocdtfntdcopcgAnstattpftihs

Sc(1aiatyHm

nc

stttgaaobsapipLemaivo(scfrpmm

A

pc0Dtmm

S. Hortal et al. / Journal of B

ally for sporocarp population studies (Martin et al.,998). Furthermore, in a critical view of this technique,eising et al. (1995) observed that the PCR condi-

ions, especially the annealing temperature, influencedhe quality of the unexpected banding patterns withscherichia coli DNA template and suggested that theajority of bands were generated by mismatch prim-

ng in a way similar to random amplified polymorphicNAs.The SSCP analysis of the specific rDNA ITS region

f L. deliciosus obtained by specific PCR amplifi-ations with the primers ITS1/LDITS2R allowed toistinguish up to nine different SSCP patterns amonghe 18 isolates tested. The alignments of the amplifiedragments of all the isolates showed up to eight singleucleotide polymorphisms (SNPs), which were poten-ially responsible for the SSCP pattern variability. Sinceivergences in the nucleotide sequence are not alwaysorrelated with differences in electrophoretic mobilityf DNA strands (Rubio et al., 1996) we identified fouroints in which single point mutations (mostly basehanges) produced conformation differences in the sin-le strands clearly detectable in the banding pattern.lthough this source of variation seems to be low, theumber of bands (two in dikarions with homozygoticequences and up to four in heterozygotic sequences:he sense and the antisense strands of the ‘normal’ allelend the altered mobility sense and antisense strands ofhe ‘variant’ allele) as well as the clear banding pat-ern make this technique very useful for tracking theersistence of a particular isolate. Moreover, samplesrom mycorrhizas and rhizospheric soil showed iden-ical patterns as those obtained from the inoculatedsolates. This is the first time that the SSCP techniqueas been applied to identify mycorrhizas and rhizo-pheric mycelium at isolate level.

On the other hand, the mobility of the strands in theSCP analysis is very sensitive to the electrophoresisonditions such as the temperature, voltage or gel typeOrita et al., 1989; Savov et al., 1992; Rubio et al.,996; Humphries et al., 1997). Thus, different poly-crylamide gels showed variable patterns for a givensolate but the patterns in the same gel are comparablend the differences remain. Also, it has been reported

hat the optimal size of the fragment for the SSCP anal-sis is between 150 and 200 bp (Savov et al., 1992;umphries et al., 1997). Although our amplified frag-ent was considerably larger (around 500 bp) the tech-

SaSa

ology 126 (2006) 123–134 131

ique maintained its sensitivity and most single-basehanges originated different, distinguishable patterns.

DNA-based identification methods provide newcopes for fine-scale studies of the dynamic spatial andemporal distribution of the mycorrhizal networks inhe soil. From the published studies, it seems likelyhat the new mycelial view of the ectomycorrhizal fun-al community will be different from both fruitbodynd root tip views (Horton and Bruns, 2001; Koide etl., 2005; Landeweert et al., 2005). From the resultsbtained in this work, tracking of the fungal sym-iont in plants inoculated with L. deliciosus in differenttages of the mycorrhiza development is viable, botht specific and intraspecific level, with relatively sim-le molecular techniques. Specific ITS-SSCP analysiss an efficient and inexpensive technique with a highotential in studies of the persistence of introduced. deliciosus isolates and its competence with nativectomycorrhizal fungi, especially at the extraradicalycelium stage. Although most on the currently avail-

ble sequence information of ectomycorrhizal fungis about the rDNA ITS region, the low intraspecificariability of the ITS sequences as well as the lackf information about the molecular basis of the SSCPWelsh et al., 1997) makes it difficult to predict theuccess of the proposed technique for the intraspecificharacterization of other fungal species. In this way,urther studies have to be undertaken on alternativeegions for the analysis of single nucleotide polymor-hisms (SNPs) and the development of microsatellitearkers to be applied in the fine monitoring of managedycorrhizal systems.

cknowledgements

The authors wish to acknowledge the financial sup-ort provided by the Instituto Nacional de Investiga-iones Agroalimentarias (INIA, Spain), projects SC00-02-C2 and RTA04-029, and the European Regionalevelopment Fund. The work is part of the doc-

orate of the first author financed by the Departa-ent d’Universitats, Recerca i Societat de la Infor-acio de la Generalitat de Catalunya and the European

ocial Fund. Technical advice of Ramon Seminagond Amaya Amador from the Unitat de Genomica,erveis Cientificotecnics, Universitat de Barcelona islso appreciated.

1 iotechn

R

A

A

A

B

B

B

C

D

D

F

G

G

G

G

G

G

G

G

G

H

H

H

H

H

H

H

H

H

H

K

L

32 S. Hortal et al. / Journal of B

eferences

lbee, S.R., Mueller, G.M., Kropp, B.R., 1996. Polymorphisms inthe large intergenic spacer of the nuclear ribosomal repeat iden-tify Laccaria proxima strains. Mycologia 88, 970–976.

micucci, A., Potenza, L., Guidi, C., Rossi, I., Bertini, L., Zam-bonelli, A., Stocchi, V., 2001. Molecular techniques for the iden-tification of edible mycorrhizal mushrooms. In: Hall, I., Yun,W., Danell, E., Zambonelli, A. (Eds.), Edible mycorrhizal mush-rooms and their cultivation. Proceedings of the Second Interna-tional Conference on Edible Mycorrhizal Mushrooms. 3–6 July,New Zealand.

micucci, A., Zambonelli, A., Giomaro, G., Potenza, L., Stocchi, V.,1998. Identification of ectomycorrhizal fungi of the genus Tuberby species-specific ITS primers. Mol. Ecol. 7, 273–277.

eidler, L.L., Hilliard, P.R., Rill, R.L., 1982. Ultrasensitive stainingof nucleic acids with silver. Anal. Biochem. 126, 374–380.

onello, P., Bruns, T.D., Gardes, M., 1998. Genetic structure of a nat-ural population of the ectomycorrhizal fungus Suillus pungens.New Phytol. 138, 533–542.

runs, T.D., White, T.J., Taylor, J.W., 1991. Fungal molecular sys-tematics. Annu. Rev. Ecol. Syst. 22, 525–564.

hen, D., Cairney, J.W.G., 2002. Investigation of the influence ofprescribed burning on ITS profiles of ectomycorrhizal and othersoil fungi at three Australian sclerophyll forest sites. Mycol. Res.106, 532–540.

i Battista, C., Bouchard, D., Martin, F., Genere, B., Amirault, J.M.,Le Tacon, F., 2002. Survival after outplanting of the ectomyc-orrhizal fungus Laccaria bicolor S238N inoculated on Douglasfir (Pseudotsuga menziesii (Mirb.) Franco) cuttings. Ann. ForestSci. 59, 81–92.

ickie, I.A., Xu, B., Koide, R., 2002. Vertical niche differentiationof ectomycorrhizal hyphae in soil as shown by T-RFLP analysis.New Phytol. 156, 527–535.

AO, 2004. Wild edible fungi. A global overview of their use andimportance to people. In: Boa, E. (Ed.), Non-wood forest prod-ucts, No. 17, Rome.

ardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificityfor basidiomycetes—application to the identification of mycor-rhizae and rusts. Mol. Ecol. 2, 113–118.

ardes, M., White, T.J., Fortin, J.A., Bruns, T.D., Taylor, J.W., 1991.Identification of indigenous and introduced symbiotic fungi inectomycorrhizae by amplification of nuclear and mitochondrialribosomal DNA. Can. J. Bot. 69, 180–190.

omes, E.A., Kasuya, M.C.M., de Barros, E.G., Borges, A.C.,Araujo, E.F., 2002. Polymorphism in the internal transcribedspacer (ITS) of the ribosomal DNA of 26 isolates of ectomy-corrhizal fungi. Genet. Mol. Biol. 25, 477–483.

uerin-Laguette, A., Conventi, S., Ruiz, G., Plassard, C., Mousain,D., 2003. The ectomycorrhizal symbiosis between Lactariusdeliciosus and Pinus sylvestris in forest soil samples: symbioticefficiency and development on roots of a rDNA internal tran-

scribed spacer-selected isolate of L. deliciosus. Mycorrhiza 13,17–25.

uerin-Laguette, A., Plassard, C., Mousain, D., 2000. Effects ofexperimental conditions on mycorrhizal relationships betweenPinus sylvestris and Lactarius deliciosus and unprecedented

L

ology 126 (2006) 123–134

fruit-body formation of the Saffron milk cap under controlledsoilless conditions. Can. J. Microbiol. 46, 790–799.

uidot, A., Debaud, J.-C., Effosse, A., Marmeisse, R., 2003. Below-ground distribution and persistence of an ectomycorrhizal fun-gus. New Phytol. 161, 539–547.

uidot, A., Debaud, J.-C., Marmeisse, R., 2002. Spatial distribu-tion of the below-ground mycelia of an ectomycorrhizal fungusinferred from specific quantification of its DNA in soil samples.FEMS Microbiol. Ecol. 42, 477–486.

uidot, A., Lumini, E., Debaud, J.-C., Marmeisse, R., 1999. Thenuclear ribosomal DNA intergenic spacer as a target sequenceto study intraspecific diversity of the ectomycorrhizal basid-iomycete Hebeloma cylindrosporum directly on Pinus root sys-tems. Appl. Environ. Microb. 65, 903–909.

uinberteau, J., Ducamp, M., Poitou, N., Mamoun, M., Olivier, J.M.,1989. Ecology of various competitors from an experimental plotof Pinus pinaster inoculated with Suillus granulatus and Lactar-ius deliciosus. Agric. Ecosyst. Environ. 28, 161–165.

all, I.R., Wang, Y., 1998. Methods for cultivating edible ectomy-corrhizal mushrooms. In: Varma, A. (Ed.), Mycorrhiza Manual.Springer-Verlag, Berlin, Heidelberg, New York, pp. 99–114.

all, I.R., Yun, W., Amicucci, A., 2003. Cultivation of edible ecto-mycorrhizal mushrooms. Trends Biotechnol. 1, 433–438.

all, T.A., 1999. BioEdit: a user-friendly biological sequence align-ment editor and analysis program for Windows 95/98/NT.Nucleic Acids Sym. Ser. 41, 95–98.

ayashi, K., 1991. PCR-SSCP: a simple and sensitive method fordetection of mutations in the genomic DNA. PCR Methods Appl.1, 34–38.

ayashi, K., 1992. PCR-SSCP: a method for detection of mutations.Genet. Anal. Tech. Appl. 3, 73–79.

enrion, B., Le Tacon, F., Martin, F., 1992. Rapid identification ofgenetic variation of ectomycorrhizal fungi by amplification ofribosomal RNA genes. New Phytol. 122, 289–298.

orton, T.R., 2002. Molecular approaches to ectomycorrhizal diver-sity studies: variation in ITS at a local scale. Plant Soil 244,29–39.

orton, T.R., Bruns, T.D., 2001. The molecular revolution in ecto-mycorrhizal ecology: peeking into the black-box. Mol. Ecol. 10,1855–1871.

umphries, S.E., Gudnason, V., Whittall, R., Day, I.N.M.,1997. Single-strand conformation polymorphism analysis withthroughput modifications, and its use in mutation detection infamilial hypercholesterolemia. Clin. Chem. 43, 427–435.

utchison, L.J., 1999. Lactarius. In: Cairney, J.W.G., Chambers,S.M. (Eds.), Ectomycorrhizal Fungi: Key Genera in Profile.Springer-Verlag, Berlin, Germany, pp. 269–285.

oide, R.T., Xu, B., Sharda, J., 2005. Contrasting below-groundviews of an ectomycorrhizal fungal community. New Phytol. 166,251–262.

andeweert, R., Leeflang, P., Kuyper, T.W., Hoffland, E., Rosling, A.,Wernars, K., Smit, E., 2003a. Molecular identification of ecto-

mycorrhizal mycelium in soil horizons. Appl. Environ. Microb.69, 327–333.

andeweert, R., Leeflang, P., Smit, E., Kuyper, T., 2005. Diversity ofan ectomycorrhizal fungal community studied by a root tip andtotal soil DNA approach. Mycorrhiza 15, 1–6.

iotechn

L

L

L

L

M

M

M

M

M

M

M

M

M

M

N

O

O

P

P

R

R

R

S

S

S

S

S

S

S. Hortal et al. / Journal of B

andeweert, R., Veenman, C., Kuyper, T.W., Fritze, H., Wernars, K.,Smit, E., 2003b. Quantification of ectomycorrhizal mycelium insoil by real-time PCR compared to conventional quantificationtechniques. FEMS Microbiol. Ecol. 45, 283–292.

anfranco, L., Perotto, S., Longato, S., Mello, A., Cometti, V., Bon-fante, P., 1998. Molecular approaches to investigate biodiversityin mycorrhizal fungi. In: Varma, A. (Ed.), Mycorrhiza Man-ual. Springer-Verlag, Berlin, Heidelberg, New York, pp. 353–372.

eake, J., Johnson, D., Donnelly, D., Muckle, G., Boddy, L., Read,D., 2004. Networks of power and influence: the role of myc-orrhizal mycelium in controlling plant communities and agroe-cosystem functioning. Can. J. Bot. 82, 1016–1045.

e Tacon, F., Alvarez, I.F., Bouchard, D., Henrion, B., Jackson,R.M., Luff, S., Parlade, J., Pera, J., Stenstrom, E., Villeneuve,N., Walker, C., 1992. Variations in field response of forest treesto nursery ectomycorrhizal inoculation in Europe. In: Read, D.J.,Lewis, D.H., Fitter, A.H., Alexander, I.J. (Eds.), Mycorrhizas inEcosystems. CAB International, Wallingford, pp. 119–134.

abru, D., Douet, J.P., Mouton, A., Dupre, C., Ricard, J.M., Med-ina, B., Castroviejo, M., Chevalier, G., 2004. PCR-RFLP using aSNP on the mitochondrial Lsu-rDNA as an easy method to differ-entiate Tuber melanosporum (Perigord truffle) and other trufflespecies in cans. Int. J. Food Microbiol. 94, 33–42.

abru, D., Dupre, C., Douet, J.P., Leroy, P., Ravel, C., Ricard, J.M.,Medina, B., Castroviejo, M., Chevalier, G., 2001. Rapid molecu-lar typing method for the reliable detection of Asiatic black truffle(Tuber indicum) in commercialized products: fruiting bodies andmycorrhizal seedlings. Mycorrhiza 11, 89–94.

artin, F., Costa, G., Delaruelle, C., Diez, J., 1998. Genomic fin-gerprinting of ectomycorrhizal fungi by microsatellite-primedPCR. In: Varma, A. (Ed.), Mycorrhiza Manual. Springer-Verlag,Berlin, Heidelberg, New York, pp. 463–474.

ello, A., Garnero, L., Bonfante, P., 1999. Specific PCR-primers asa reliable tool for the detection of white truffles in mycorrhizalroots. New Phytol. 141, 511–516.

ello, A., Ghignone, S., Vizzini, A., Sechi, C., Ruiu, P., Bonfante,P., 2006. ITS primers for the identification of marketable boletes.J. Biotechnol. 121, 318–329.

eyer, W., Mitchell, T.G., Freedman, E.Z., Vilgalys, R., 1993.Hybridization probes for conventional DNA fingerprinting usedas single primers in the polymerase chain reaction to distin-guish strains of Cryptococcus neoformans. J. Clin. Microbiol.31, 2274–2280.

ohabeer, A.J., Hiti, A.L., Martin, W.J., 1991. Non-radioactivesingle-strand conformation polymorphism (SSCP) using thePharmacia ‘Phast-System’. Nucleic Acids Res. 19, 3154.

olina, R., 1979. Pure culture synthesis and host specificity of redalder mycorrhizae. Can. J. Bot. 57, 1223–1228.

olina, R., Palmer, J.G., 1982. Isolation, maintenance, and pure cul-ture manipulation of ectomycorrhizal fungi. In: Schenk, N.C.(Ed.), Methods and Principles of Mycorrhizal Research. Ameri-

can Phytopathology Society, St. Paul, MN, pp. 115–129.

urat, C., Vizzini, A., Bonfante, P., Mello, A., 2005. Morphologicaland molecular typing of the below-ground fungal community in anatural Tuber magnatum truffle-ground. FEMS Microbiol. Lett.,307–313.

S

ology 126 (2006) 123–134 133

air, S., Lin, T.K., Pang, T., Altwegg, M., 2002. Characterization ofSalmonella serovars by PCR-single strand conformation poly-morphism analysis. J. Clin. Microbiol. 40, 2346–2351.

ort, A.J.P., 1981. Nutritional requirements of Lactarius species andcultural characters in relation to taxonomy. Ver. Km. Ned. Akad.Wet. Natuur 76, 1–95.

rita, M., Iwahana, H., Kanazawa, H., Hayashi, K., Sekiya, T., 1989.Detection of polymorphisms of human DNA by gel electrophore-sis as single-strand conformation polymorphisms. Proc. Natl.Acad. Sci. U.S.A. 86, 2766–2770.

arlade, J., Pera, J., Luque, J., 2004. Evaluation of mycelial inocula ofedible Lactarius species for the production of Pinus pinaster andP. sylvestris mycorrhizal seedlings under greenhouse conditions.Mycorrhiza 14, 171–176.

oitou, N., Mamoun, M., Ducamp, M., Delmas, J., 1984. Apres lebolet granuleux, le Lactaire delicieux obtenu en fructification auchamp a partir de plants mycorhizes. PHM Rev. Hortic. 244,65–68.

incon, A., Alvarez, I.F., Pera, J., 1999. Ectomycorrhizal fungiof Pinus pinea L. in northeastern Spain. Mycorrhiza 8, 271–276.

osling, A., Landeweert, R., Lindahl, B.D., Larsson, K.-H., Kuyper,T.W., Taylor, A.F.S., Finlay, R.D., 2003. Vertical distribution ofectomycorrhizal fungal taxa in a podzol soil profile. New Phytol.159, 775–783.

ubio, L., Ayllon, M.A., Guerri, J., Pappu, H., Niblett, C., Moreno,P., 1996. Differentiation of citrus tristeza closterovirus (CTV)isolates by single-strand conformation polymorphism analy-sis of the coat protein gene. Ann. Appl. Biol. 129, 479–489.

ambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning:A Laboratory Manual, 2nd ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York, USA.

avov, A., Angelicheva, D., Jordanova, A., Eigel, A., Kalaydjieva,L., 1992. High percentage acrylamide gels improve resolution inSSCP analysis. Nucleic Acids Res. 20, 6741–6742.

chubert, R., Raidl, S., Funk, R., Bahnweg, G., Muller-Starck, G.,Agerer, R., 2003. Quantitative detection of agar-cultivated andrhizotron-grown Piloderma croceum Erikss & Hjortst. by ITS1-based fluorescent PCR. Mycorrhiza 13, 159–165.

ejalon-Delmas, N., Roux, C., Martins, M., Kulifaj, M., Becard,G., Dargent, R., 2000. Molecular tools for the identification ofTuber melanosporum in agro industry. J. Agric. Food Chem. 48,2608–2613.

elosse, M.A., Costa, G., Di Battista, C., Le Tacon, F., Martin, F.,1996. Meiotic segregation and recombination of the intergenicspacer of the ribosomal DNA in the ectomycorrhizal basid-iomycete Laccaria bicolor. Curr. Genet. 30, 332–337.

elosse, M.A., Jacquot, D., Bouchard, D., Martin, F., Le Tacon,F., 1998. Temporal persistence and spatial distribution of anAmerican inoculant strain of the ectomycorrhizal basidiomyceteLaccaria bicolor in a French forest plantation. Mol. Ecol. 7,

561–573.

elosse, M.A., Martin, F., Bouchard, D., Le Tacon, F., 1999. Structureand dynamics of experimentally introduced and naturally occur-ring Laccaria sp. discrete genotypes in a Douglas-fir plantation.Appl. Environ. Microb. 65, 2006–2014.

1 iotechn

S

S

S

T

V

W

W

W

W

34 S. Hortal et al. / Journal of B

elosse, M.A., Martin, F., Le Tacon, F., 2001. Intraspecific variationin fruiting phenology in an ectomycorrhizal Laccaria populationunder Douglas-fir. Mycol. Res. 105, 524–531.

inger, R., 1986. The Agaricales in Modern Taxonomy, 4th ed. KoeltzScientific Books, Koenigstein, Germany, 981 pp.

tenlid, J., Karlsson, J.O., Hogberg, N., 1994. Intraspecificgenetic variation in Heterobasidion annosum revealed byamplification of minisatellite DNA. Mycol. Res. 99, 179–186.

rappe, J.M., 1962. Fungus associates of ectotrophic mycorrhizae.Bot. Rev. 28, 538–606.

illeneuve, N., Le Tacon, F., Bouchard, D., 1991. Survival of inoc-ulated Laccaria bicolor in competition with native ectomycor-

rhizal fungi and effects on the growth of outplanted Douglas-firseedlings. Plant Soil 135, 95–107.

allander, H., Mahmood, S., Hagerberg, D., Johansson, L., Pal-lon, J., 2003. Elemental composition of ectomycorrhizal myceliaidentified by PCR-RFLP analysis and grown in contact with

W

ology 126 (2006) 123–134

apatite or wood ash in forest soil. FEMS Microbiol. Ecol. 44,57–65.

eber, J., Diez, J., Selosse, M.A., Tagu, D., Le Tacon, F., 2002.SCAR markers to detect mycorrhizas of an American Laccariabicolor strain inoculated in European Douglas-fir plantations.Mycorrhiza 12, 19–27.

eising, K., Atkinson, R.G., Gardner, R.C., 1995. Genomic finger-printing by microsatellite-primed PCR: a critical evaluation. PCRMethods Appl. 4, 249–255.

elsh, J.A., Castren, K., Vahakangas, K.H., 1997. Single-strandconformation polymorphism analysis to detect p53 mutations:characterization and development of controls. Clin. Chem. 43,2251–2255.

hite, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification anddirect sequencing of fungal ribosomal RNA genes for phyloge-netics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J.(Eds.), PCR protocols. A guide to Methods and Applications.Academic Press, San Diego, CA, pp. 315–322.